CN109486868B - Method for producing isopropanol and butanol by fermenting lignocellulose serving as raw material - Google Patents
Method for producing isopropanol and butanol by fermenting lignocellulose serving as raw material Download PDFInfo
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- CN109486868B CN109486868B CN201710808876.2A CN201710808876A CN109486868B CN 109486868 B CN109486868 B CN 109486868B CN 201710808876 A CN201710808876 A CN 201710808876A CN 109486868 B CN109486868 B CN 109486868B
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- Prior art keywords
- fermentation
- culture medium
- concentration
- isopropanol
- butanol
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- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 title claims abstract description 117
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 title claims abstract description 92
- 239000002994 raw material Substances 0.000 title claims abstract description 20
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 103
- 230000004151 fermentation Effects 0.000 claims abstract description 98
- 239000001963 growth medium Substances 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 34
- 239000002609 medium Substances 0.000 claims abstract description 31
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000001913 cellulose Substances 0.000 claims abstract description 20
- 229920002678 cellulose Polymers 0.000 claims abstract description 20
- 238000004880 explosion Methods 0.000 claims abstract description 20
- 241000193454 Clostridium beijerinckii Species 0.000 claims abstract description 19
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 17
- 238000002360 preparation method Methods 0.000 claims abstract description 17
- 235000015097 nutrients Nutrition 0.000 claims abstract description 11
- 239000000463 material Substances 0.000 claims abstract description 8
- 150000003839 salts Chemical class 0.000 claims abstract description 8
- 229920002488 Hemicellulose Polymers 0.000 claims abstract description 7
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 6
- 229940088594 vitamin Drugs 0.000 claims abstract description 5
- 239000011782 vitamin Substances 0.000 claims abstract description 5
- 235000013343 vitamin Nutrition 0.000 claims abstract description 5
- 229930003231 vitamin Natural products 0.000 claims abstract description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 3
- 230000001502 supplementing effect Effects 0.000 claims abstract description 3
- 239000007788 liquid Substances 0.000 claims description 37
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 235000002639 sodium chloride Nutrition 0.000 claims description 19
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- 240000008042 Zea mays Species 0.000 claims description 14
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 14
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 14
- 235000005822 corn Nutrition 0.000 claims description 14
- 239000010902 straw Substances 0.000 claims description 14
- 235000015278 beef Nutrition 0.000 claims description 13
- 239000000284 extract Substances 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 13
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 12
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 12
- 108010059892 Cellulase Proteins 0.000 claims description 12
- 239000001888 Peptone Substances 0.000 claims description 12
- 108010080698 Peptones Proteins 0.000 claims description 12
- 229940106157 cellulase Drugs 0.000 claims description 12
- 235000019319 peptone Nutrition 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 claims description 10
- 238000011218 seed culture Methods 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 9
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 9
- 239000011790 ferrous sulphate Substances 0.000 claims description 9
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 9
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 9
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 8
- 239000001110 calcium chloride Substances 0.000 claims description 8
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 8
- 235000011148 calcium chloride Nutrition 0.000 claims description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 8
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 8
- 239000008399 tap water Substances 0.000 claims description 8
- 235000020679 tap water Nutrition 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 238000001784 detoxification Methods 0.000 claims description 7
- 230000007935 neutral effect Effects 0.000 claims description 7
- 235000010469 Glycine max Nutrition 0.000 claims description 6
- 244000068988 Glycine max Species 0.000 claims description 6
- 229960002685 biotin Drugs 0.000 claims description 6
- 235000020958 biotin Nutrition 0.000 claims description 6
- 239000011616 biotin Substances 0.000 claims description 6
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 6
- 235000010216 calcium carbonate Nutrition 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 229920005610 lignin Polymers 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 238000011065 in-situ storage Methods 0.000 claims description 5
- 239000011691 vitamin B1 Substances 0.000 claims description 5
- 125000001477 organic nitrogen group Chemical group 0.000 claims description 4
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 4
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 claims description 3
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 3
- 239000005695 Ammonium acetate Substances 0.000 claims description 3
- 241000196324 Embryophyta Species 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 229960004050 aminobenzoic acid Drugs 0.000 claims description 3
- 235000019257 ammonium acetate Nutrition 0.000 claims description 3
- 229940043376 ammonium acetate Drugs 0.000 claims description 3
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 claims description 3
- 239000000920 calcium hydroxide Substances 0.000 claims description 3
- 229910001861 calcium hydroxide Inorganic materials 0.000 claims description 3
- 229960001305 cysteine hydrochloride Drugs 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- 235000011121 sodium hydroxide Nutrition 0.000 claims description 3
- 108010009736 Protein Hydrolysates Proteins 0.000 claims description 2
- 235000019764 Soybean Meal Nutrition 0.000 claims description 2
- 229930003451 Vitamin B1 Natural products 0.000 claims description 2
- 230000003213 activating effect Effects 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- 239000007789 gas Substances 0.000 claims description 2
- 230000007062 hydrolysis Effects 0.000 claims description 2
- 238000006460 hydrolysis reaction Methods 0.000 claims description 2
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 claims description 2
- 229910000360 iron(III) sulfate Inorganic materials 0.000 claims description 2
- 229940099596 manganese sulfate Drugs 0.000 claims description 2
- 239000011702 manganese sulphate Substances 0.000 claims description 2
- 235000007079 manganese sulphate Nutrition 0.000 claims description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 238000010298 pulverizing process Methods 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 239000004317 sodium nitrate Substances 0.000 claims description 2
- 235000010344 sodium nitrate Nutrition 0.000 claims description 2
- 239000004455 soybean meal Substances 0.000 claims description 2
- 229960003495 thiamine Drugs 0.000 claims description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 2
- 235000010374 vitamin B1 Nutrition 0.000 claims description 2
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 2
- 239000002023 wood Substances 0.000 claims description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 56
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 28
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 28
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 22
- 239000008103 glucose Substances 0.000 description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 21
- 239000000243 solution Substances 0.000 description 18
- 238000001514 detection method Methods 0.000 description 16
- 241000894006 Bacteria Species 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 239000000047 product Substances 0.000 description 7
- 229920001221 xylan Polymers 0.000 description 7
- 150000004823 xylans Chemical class 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 229920001542 oligosaccharide Polymers 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 241000193401 Clostridium acetobutylicum Species 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- 241000193403 Clostridium Species 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- JVBXVOWTABLYPX-UHFFFAOYSA-L sodium dithionite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])=O JVBXVOWTABLYPX-UHFFFAOYSA-L 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000011575 calcium Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000009736 wetting Methods 0.000 description 3
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 108060008225 Thiolase Proteins 0.000 description 2
- 102000002932 Thiolase Human genes 0.000 description 2
- 102000004357 Transferases Human genes 0.000 description 2
- 108090000992 Transferases Proteins 0.000 description 2
- 108091022873 acetoacetate decarboxylase Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000012459 cleaning agent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 241001112696 Clostridia Species 0.000 description 1
- 241000193171 Clostridium butyricum Species 0.000 description 1
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 description 1
- 108010001817 Endo-1,4-beta Xylanases Proteins 0.000 description 1
- 230000002528 anti-freeze Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 101150054092 buk gene Proteins 0.000 description 1
- DNZWLJIKNWYXJP-UHFFFAOYSA-N butan-1-ol;propan-2-one Chemical compound CC(C)=O.CCCCO DNZWLJIKNWYXJP-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 239000013064 chemical raw material Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000012024 dehydrating agents Substances 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 108010084715 isopropanol dehydrogenase (NADP) Proteins 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/16—Butanols
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for producing isopropanol and butanol by fermenting lignocellulose as a raw material, which comprises the following steps of firstly carrying out steam explosion pretreatment on the lignocellulose raw material at the temperature of 120-160 ℃ and the pressure of 0.8-1.3 MPa; carrying out enzymolysis on the pretreated materials to obtain a cellulose and hemicellulose hydrolyzed mixed sugar solution; then, taking the hydrolyzed mixed sugar solution as a carbon source, and supplementing a nitrogen source, nutrient salts and vitamins to prepare a fermentation medium; inoculating the seed solution of clostridium beijerinckii XH0906 into a fermentation culture medium, and fermenting to produce butanol and isopropanol. The method firstly carries out steam explosion pretreatment on the lignocellulose raw material, and then utilizes clostridium beijerinckii XH0906 to ferment and produce the butanol and the isopropanol, and has the characteristics of simple preparation process, high fermentation yield and the like.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for producing isopropanol and butanol by fermentation by using lignocellulose as a raw material.
Background
Butanol is an important organic chemical raw material and has wide application in the industrial departments of chemical industry, medicine, petroleum and the like. Butanol has two more methylene groups than ethanol, has higher hydrophobicity and lower volatility than ethanol, can be mixed with gasoline in any proportion, and has a calorific value equivalent to that of gasoline. Butanol is receiving increasing attention from countries around the world as a potential renewable bio-energy source that can replace gasoline.
Isopropanol is an important chemical product and raw material. It is mainly used as dehydrating agent and cleaning agent in pharmacy, cosmetics, plastics, perfume, paint and electronic industry. Isopropanol is widely used in the production of industrial and consumer products as a low cost solvent or extractant. Low quality isopropyl alcohol can also be used in automotive fuels. In many cases isopropanol can be used instead of ethanol. The total annual demand of China in 2010 for the isopropanol reaches 30 ten thousand tons, and the isopropanol mainly serves as a solvent or an extractant in the processes of printing ink, coating and pharmaceutical industry, and the consumption amount of the isopropanol accounts for about 60 percent of the total consumption amount of the isopropanol. In the field of chemical intermediates, isopropanol is mainly used for producing isopropylamine, isopropyl ether and esters in China, and the consumption amount of the isopropanol accounts for about 25 percent of the total consumption amount of the isopropanol. Other applications of the isopropanol mainly comprise electronic industry cleaning agents, automobile antifreeze solutions, disinfectants, washing products, daily chemical products and the like, and the consumption amount of the isopropanol accounts for about 15 percent of the total consumption amount of the isopropanol.
CN105713823A discloses a device and a method for producing isopropanol and butanol by extraction fermentation, which comprises a common bioreactor, a fiber bed immobilized reactor and a liquid-liquid extractor which are connected in series by pipelines in sequence; the fiber bed immobilization reactor is connected with a common bioreactor through a return pipeline to form a first liquid circulation system; the extractor is connected with the bioreactor through a return pipeline to form a second liquid circulation system. Firstly, culturing seed liquid of the alcohol-producing bacteria in a bioreactor, fixing the bacteria in a fiber bed immobilized reactor when the seed liquid reaches the middle stage of logarithmic growth, fermenting until the seed liquid enters an alcohol-producing stage, introducing fermentation liquor into an extractor for extraction, and finally refluxing to the bioreactor. The alcohol-producing strain of the invention is isopropanol and butanol or all anaerobic clostridia producing butanol, and is not particularly limited. Strains are exemplified by Clostridium beijerinckii, Clostridium acetobutylicum, Clostridium glycollate and Clostridium butyricum. But in practice not any clostridium beijerinckii can co-produce isopropanol and butanol under normal conditions.
CN101688202 discloses an isopropanol producing bacterium which is imparted with acetoacetate decarboxylase activity, isopropanol dehydrogenase activity, CoA transferase activity and thiolase activity and can produce isopropanol from a plant-derived material. The bacterium is Escherichia coli, and the gene is connected with glyceraldehyde-3-phosphate dehydrogenase promoter, wherein, the activity of the acetoacetate decarboxylase is obtained by introducing the gene coding the enzyme from Clostridium acetobutylicum, the activity of the isopropanol dehydrogenase is obtained by introducing the gene coding the enzyme from Clostridium beijerinckii, and the activity of CoA transferase and thiolase is obtained by introducing the gene coding the enzyme from Escherichia coli or Clostridium acetobutylicum.
CN 102161979A discloses a recombinant bacterium for co-producing butanol, isopropanol and ethanol and application thereof, which is 8 kinds of recombinant bacteria constructed by genetic engineering means, wherein the producing bacterium A is Clostridium (Clostridium) for producing acetone butanol; the outbreak B is Clostridium acetobutylicum (Clostridium) with a buk gene knocked out.
CN102199614A discloses an engineering bacterium for stably co-producing isopropanol and butanol, a construction method and application thereof. The construction method of the engineering bacteria comprises the following steps: and integrating the encoding gene of the secondary alcohol dehydrogenase into the genome of the clostridium acetobutylicum to obtain the recombinant bacterium, namely the engineering bacterium. The engineering bacteria can stably produce butanol, isopropanol and ethanol.
The method for producing isopropanol and butanol is basically realized by using engineering bacteria constructed by genetic engineering, the construction process is complex, and the product yield is to be improved. In addition, a certain amount of ethanol is generated, and the effect is to be improved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for producing isopropanol and butanol by fermenting lignocellulose serving as a raw material. The method firstly carries out steam explosion pretreatment on the lignocellulose raw material, and then utilizes clostridium beijerinckii XH0906 to ferment and produce the butanol and the isopropanol, and has the characteristics of simple preparation process, high fermentation yield and the like.
The invention discloses a method for producing isopropanol and butanol by fermenting lignocellulose as a raw material, which comprises the following steps:
(1) performing steam explosion pretreatment on the lignocellulose raw material at the temperature of 160 ℃ at 120 ℃ and the pressure of 0.8-1.3 MPa;
(2) carrying out enzymolysis on the pretreated material to obtain a cellulose and hemicellulose hydrolyzed mixed sugar solution;
(3) preparing a fermentation medium by taking the hydrolyzed mixed sugar solution as a carbon source and supplementing a nitrogen source, nutrient salts and vitamins;
(4) inoculating the seed solution of clostridium beijerinckii XH0906 into the fermentation medium in the step (3), and fermenting to produce butanol and isopropanol.
The lignocellulose raw material in the step (1) is straw, wood chips, energy plants and the like containing cellulose, hemicellulose and lignin, preferably straw, and further preferably corn straw.
And (2) performing neutral steam explosion pretreatment on the steam explosion in the step (1). The specific process is as follows: pulverizing the lignocellulose raw material to 0.5-5 cm, adding tap water with the mass of 2-4 times, soaking, introducing into a detention device of a steam explosion device, maintaining for 5-20min at the temperature of 120-.
Preparing the pretreated material in the step (1) into feed liquid with a solid-liquid ratio of 8-20% (w/v), wherein the solid-liquid ratio refers to the percentage (g: mL) of solid mass to liquid volume, and then adding a cellulase preparation for enzymolysis. The cellulase preparation is any enzyme protein or enzyme protein mixture capable of hydrolyzing cellulose and hemicellulose into monosaccharide, wherein the enzyme protein or enzyme protein mixture at least comprises 4 types of enzyme protein components: cellulose exoenzyme, cellulose endoenzyme, beta-glucosidase and xylanase. The cellulase may be a commercial enzyme or produced in situ by fermentation using an enzyme-producing strain. The addition amount of cellulase is 20-50FPIU/g cellulose, the pH value of enzymolysis is 4.5-5.5, the temperature is 45-55 ℃, the stirring speed is 50-300r/min, and the enzymolysis time is 24-72 h.
And (3) directly using the hydrolyzed mixed sugar solution obtained in the step (2) for preparation of a fermentation medium without solid-liquid separation and detoxification processes. The amount of the mixed sugar solution for hydrolysis is such that the sugar concentration in the medium is 30 to 60g/L, preferably 40 to 50 g/L.
The nitrogen source supplemented in the step (3) is an organic nitrogen source or/and an inorganic nitrogen source. The organic nitrogen source can be one or more of yeast extract, peptone, beef extract, corn steep liquor, soybean meal hydrolysate, etc., preferably peptone and beef extract, with concentration of 0.1-20 g/L. The inorganic nitrogen source can be one or more of ammonium acetate, sodium nitrate, ammonium sulfate, etc., preferably ammonium sulfate, with a concentration of 0.05-5 g/L. More preferred nitrogen sources are soy peptone 8-12g/L, beef extract 0.4-0.8g/L and ammonium sulfate 0.6-1.2 g/L. The supplementary nutrient salt can be one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, sodium hydroxide, ferrous sulfate, ferric sulfate, magnesium sulfate, calcium chloride, manganese sulfate, calcium carbonate and the like, and the concentration is 1-6 g/L. More preferably, the inorganic salt is 2-4 g/L potassium dihydrogen phosphate, 0.3-0.6 g/L sodium chloride, 0.05-0.2 g/L ferrous sulfate, 0.1-0.4g/L magnesium sulfate, and 0.05-0.2 g/L calcium chloride. The vitamin is one or more of vitamin B1, biotin, and p-aminobenzoic acid, and has concentration of 0.01-0.1 g/L, or not. Furthermore, 0.2-5g/L calcium carbonate is added into the fermentation medium, the pH is not controlled in the whole process, and the fermentation medium does not need to be sterilized.
The pH of the fermentation medium in step (3) is 6.0-8.0, preferably 6.5-7.5, and can be adjusted by various inorganic bases, preferably by calcium hydroxide.
Clostridium beijerinckii (of step (4)) (Clostridium beijerinckii) XH0906, accession number CGMCC number 9124, published in CN201510638879.7 and submitted preservation and proof of survival.
The seed culture medium for preparing the seed liquid in the step (4) can have the same or different components with the fermentation culture medium. The following formulation of seed culture medium is preferably employed: the dosage of the hydrolyzed mixed sugar solution is such that the sugar concentration is 2-8 g/L, peptone is 5-15 g/L, yeast extract is 2-5g/L, beef extract is 5-15 g/L, cysteine hydrochloride is 0.2-0.6 g/L, sodium chloride is 1-4 g/L, sodium acetate is 2-4 g/L, the pH is adjusted to 6.0-8.5, and sterilization is carried out at 115 ℃ for 30 min. The solid medium is prepared by adding 1wt% to 2wt% agar to a liquid medium such as a fermentation medium.
And (4) preparing the seed solution in the step (4), namely activating a clostridium beijerinckii XH0906 plate in a solid culture medium, placing the plate in an anaerobic environment, culturing for 12 to 48 hours at the temperature of between 28 and 42 ℃, then picking the activated single colony, inoculating the single colony into the seed culture medium, and standing and culturing for 12 to 48 hours at the temperature of between 28 and 42 ℃.
The fermentation process adopted in the step (4) can adopt the existing known processes, such as stirred tank fermentation, in-situ extraction fermentation, gas in-situ extraction fermentation and the like. Preferably, a stirring tank is adopted for fermentation, stirring is carried out to ensure the uniformity between the culture medium and the thalli in the fermentation system, and the stirring linear velocity is 50-300 r/min.
Inoculating the seed liquid in the step (4) into a liquid fermentation culture medium in an inoculation amount of 2-20% by volume, preferably 5-15%. The fermentation condition is anaerobic fermentation or facultative fermentation, and the fermentation temperature is 28-42 ℃, preferably 32-38 ℃; the fermentation time is 24-120h, preferably 24-72 h. Clostridium beijerinckii XH0906 is adopted as the fermentation strain, sodium hydrosulfite is not required to be added into the culture medium to remove oxygen when facultative oxygen condition is adopted, and N is not required to be introduced into the culture process2Maintaining an anaerobic environment.
Compared with the prior art, the invention has the following beneficial effects:
(1) the low-strength neutral steam explosion pretreatment process is adopted, the xylose loss rate is reduced, the concentration of inhibitors in the pretreated materials is reduced, and the hydrolysate can be used for the fermentation and coproduction of butanol and acetone of carboxyli bailii XH0906 without detoxification, so that the steps of solid-liquid separation, detoxification treatment and the like before the fermentation process are omitted, and the fermentation cost is reduced.
(2) By adopting a neutral steam explosion pretreatment process with lower strength, an inhibitor with proper concentration can be generated, which is beneficial to inducing carboxybacter bailii XH0906 to co-produce isopropanol and butanol, the butanol metabolism flux is reduced, the isopropanol metabolism flux is increased, and finally the proportion of the isopropanol and the butanol is close to 4:5-1:1, so that the fermentation effect is improved.
(3) The carboxybacter bailii XH0906 can be fermented under the facultative condition, so that the oxygen removal process in the traditional anaerobic fermentation process is reduced, and N does not need to be introduced in the whole process2The phenomenon of no growth of thalli caused by unclean deoxidization is avoided, and the energy consumption is reduced.
(4) Calcium carbonate is added into the culture medium to keep the pH value relatively stable in the fermentation process, and simultaneously, necessary calcium ions are provided for thallus metabolism, so that the yield of isopropanol is improved, and the fermentation time is shortened.
(5) The lignocellulose is adopted as the raw material to ferment and co-produce the butanol and the isopropanol, thereby reducing the consumption of grains, changing waste into valuable and simultaneously increasing the income of farmers.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, but the protection scope of the invention is not limited by the following embodiments. In the present invention, wt% is a mass fraction.
The lignocellulose raw material used in the embodiment of the invention is corn straw, wherein the cellulose accounts for 38.2wt%, the hemicellulose accounts for 22.1wt%, the lignin accounts for 20.2wt%, and the ash accounts for 3.9wt%, and the raw material is crushed to the particle size of 1-5 cm by a crusher.
The invention analyzes the product and the by-product in the fermentation liquor by a liquid chromatograph to calculate the concentration of the main components: liquid chromatograph (Agilent 1200), column chromatography is Bolete HPX-87H (300 mm × 7.8 mm), mobile phase is 0.005mol/L H2SO4The flow rate of the aqueous solution is 0.6mL/min, the column incubator is 65 ℃, the detector is a differential detector (Agilent 1200), the temperature of the detector is 45 ℃, and the sample injection amount is 5 mu L. The dry matter concentration was determined by drying with a Sidolisi HG63 moisture meter.
Glucose enzymolysis yield = hydrolyzed glucose mass × 0.9/cellulose mass × 100%; xylose enzymolysis yield = hydrolyzed xylose mass × 0.88/xylan mass × 100%.
Example 1
(1) Taking crushed corn straws, adding tap water according to a solid-to-liquid ratio of 1g:3mL for wetting, entering a detention device of a steam explosion device, maintaining at 150 ℃ and 1.0MPa for 10min, and instantaneously decompressing and exploding to obtain neutral steam explosion pretreated corn straws. Mainly consisting of xylose, xylo-oligosaccharides, xylan, cellulose and lignin, with a dry matter concentration of 32%, a xylose, xylo-oligosaccharides and xylan content of 19.11% (relative to dry matter) and a cellulose content of 39.7% (relative to dry matter).
(2) Adding tap water into the pretreated corn straws to adjust the dry matter concentration to 10%, adopting the cellulase Ctec2 of Novitin, adding the cellulase 30FPIU/g cellulose, performing enzymolysis for 72 hours at 50 ℃ and 150r/min, wherein the pH value of the enzymolysis is 5.0. Through detection, the xylose concentration is 19g/L, the glucose concentration is 39g/L, the glucose enzymolysis yield is 84%, and the xylose enzymolysis yield is 83%.
(3) The hydrolyzed mixed sugar solution is directly used for preparing a fermentation culture medium without solid-liquid separation and detoxification processes, and the dosage of the hydrolyzed mixed sugar solution is 50 g/L. The following nutrient elements are supplemented to prepare a fermentation medium: 8g/L of soybean peptone, 0.4g/L of beef extract and 1g/L of ammonium sulfate; 4g/L potassium dihydrogen phosphate, 0.4g/L magnesium sulfate, 0.2g/L ferrous sulfate, 0.6g/L sodium chloride and 0.2g/L calcium chloride; vitamin B10.04g/L, 0.04g/L para aminobenzoic acid and 0.004g/L biotin. Addition of Ca (OH)2The pH was adjusted to 7.0. Sterilization is not required. The solid medium was prepared by adding 1.5wt% agar to a liquid medium.
(4) The seed culture medium adopts the same components as the fermentation culture medium, the clostridium beijerinckii XH0906 is activated on a solid culture medium plate, is placed in an anaerobic environment and is cultured for 24 hours at the temperature of 30 ℃, then the activated single colony is selected and inoculated into the culture medium, oxygen is not required to be removed, and the culture medium is kept stand and cultured for 24 hours at the temperature of 35 ℃ to obtain the seed solution. Inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 10% of the volume ratio for fermentation, and adopting a vertical stirring tank, wherein the stirring linear speed is 150 r/min. The fermentation condition is facultative fermentation, the culture medium does not need to be added with sodium hydrosulfite for deoxidation, the fermentation temperature is 34 ℃, and N does not need to be introduced in the fermentation process2And (3) maintaining an anaerobic environment, naturally adjusting the pH value, and fermenting for 48 hours to obtain fermentation liquor containing butanol and isopropanol.
The detection proves that the concentration of the butanol in the fermentation liquor is 6.05 g/L, the concentration of the isopropanol is 5.41g/L, and the concentrations of the residual glucose and the xylose are 7.2 g/L and 5.4 g/L respectively.
Example 2
(1) Taking crushed corn straws, adding tap water according to a solid-to-liquid ratio of 1g:2mL for wetting, entering a detention device of a steam explosion device, maintaining at 120 ℃ and 1.3MPa for 20min, and instantaneously releasing pressure and exploding to obtain neutral steam explosion pretreated corn straws. Mainly consisting of xylose, xylo-oligosaccharides, xylan, cellulose and lignin, with a dry matter concentration of 32%, a xylose, xylo-oligosaccharides and xylan content of 15.86% (relative to dry matter) and a cellulose content of 42.71% (relative to dry matter).
(2) Adding tap water into the pretreated corn straws to adjust the dry matter concentration to 8%, adopting the cellulase Ctec2 of Novitin, adding 20FPIU/g of the cellulase, performing enzymolysis at 45 ℃ and 100r/min for 72 hours, wherein the pH value of the enzymolysis is 4.5. Through detection, the xylose concentration is 13g/L, the glucose concentration is 33g/L, the glucose enzymolysis yield is 84%, and the xylose enzymolysis yield is 87%.
(3) The hydrolyzed mixed sugar solution is directly used for preparing a fermentation culture medium without solid-liquid separation and detoxification processes, and the dosage of the hydrolyzed mixed sugar solution is such that the sugar concentration is 40 g/L. The following nutrient elements are supplemented to prepare a fermentation medium: 10g/L of soybean peptone, 0.6g/L of beef extract and 1.0g/L of ammonium sulfate; 3g/L potassium dihydrogen phosphate, 0.3g/L magnesium sulfate, 0.1g/L ferrous sulfate, 0.4g/L sodium chloride and 0.1g/L calcium chloride; vitamin B10.02g/L, 0.02g/L of p-aminobenzoic acid and 0.001g/L of biotin. Addition of Ca (OH)2The pH was adjusted to 6.5. Sterilization is not required. The solid medium was prepared by adding 1.5wt% agar to a liquid medium.
(4) The seed culture medium adopts the same components as the fermentation culture medium, the clostridium beijerinckii XH0906 is activated in a solid culture medium plate, is placed in an anaerobic environment and is cultured for 24 hours at 34 ℃, then the activated single colony is selected and inoculated into the culture medium, oxygen is not required to be removed, and the culture medium is kept still for 24 hours at 34 ℃, so that the seed solution is obtained. Inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 15 percent of the volume ratioFermenting with a vertical stirring tank at a stirring speed of 100 r/min. The fermentation condition is facultative fermentation, the culture medium does not need to be added with sodium hydrosulfite for deoxidation, the fermentation temperature is 34 ℃, and N does not need to be introduced in the fermentation process2And (3) maintaining an anaerobic environment, naturally adjusting the pH value, and fermenting for 48 hours to obtain fermentation liquor containing butanol and isopropanol.
The detection proves that the concentration of the butanol in the fermentation liquor is 6.01 g/L, the concentration of the isopropanol is 5.43g/L, and the concentrations of the residual glucose and the xylose are 1.7 g/L and 2.5 g/L respectively.
Example 3
(1) Taking crushed corn straws, adding tap water according to a solid-to-liquid ratio of 1g:4mL for wetting, entering a detention device of a steam explosion device, maintaining for 5min at 160 ℃ and under a pressure of 0.8MPa, and instantaneously releasing pressure and exploding to obtain the neutral steam explosion pretreated corn straws. Mainly consisting of xylose, xylo-oligosaccharides, xylan, cellulose and lignin, with a dry matter concentration of 32%, a xylose, xylo-oligosaccharides and xylan content of 20.34% (relative to dry matter) and a cellulose content of 38.57% (relative to dry matter).
(2) Adding tap water into the pretreated corn straws to adjust the dry matter concentration to 20%, adopting 50FPIU/g cellulose as cellulase Ctec2, carrying out enzymolysis at 55 ℃ and 200r/min for 72h, wherein the addition amount of the cellulase is Ctec 5.5. Through detection, the xylose concentration is 45 g/L, the glucose concentration is 73g/L, the glucose enzymolysis yield is 77%, and the xylose enzymolysis yield is 88%.
(3) The hydrolyzed mixed sugar solution is directly used for preparing a fermentation culture medium without the processes of solid-liquid separation and detoxification, and the sugar concentration of the hydrolyzed mixed sugar solution is 58 g/L. The following nutrient elements are supplemented to prepare a fermentation medium: soybean peptone 12g/L, beef extract 0.8g/L, ammonium sulfate 1.2 g/L; 2g/L potassium dihydrogen phosphate, 0.1g/L magnesium sulfate, 0.05g/L ferrous sulfate, 0.05g/L sodium chloride and 0.05g/L calcium chloride; vitamin B10.04g/L, 0.04g/L para aminobenzoic acid and 0.004g/L biotin. Addition of Ca (OH)2The pH was adjusted to 7.5. Sterilization is not required. The solid medium was prepared by adding 1.5wt% agar to a liquid medium.
(4) Seed culture medium adopts and ferments and cultivatesActivating a solid culture medium plate by using clostridium beijerinckii XH0906 with the same components, culturing for 24h at 34 ℃ in an anaerobic environment, then selecting an activated single colony to be inoculated into the culture medium without removing oxygen, and standing and culturing for 24h at 34 ℃ to obtain a seed solution. Inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 7% of the volume ratio for fermentation, and adopting a vertical stirring tank at the stirring linear speed of 200 r/min. The fermentation condition is facultative fermentation, the culture medium does not need to be added with sodium hydrosulfite for deoxidation, the fermentation temperature is 34 ℃, and N does not need to be introduced in the fermentation process2And (3) maintaining an anaerobic environment, naturally adjusting the pH value, and fermenting for 72 hours to obtain fermentation liquor containing butanol and isopropanol.
The detection proves that the concentration of the butanol in the fermentation liquor is 6.22g/L, the concentration of the isopropanol is 5.67g/L, and the concentrations of the residual glucose and the xylose are 6.3g/L and 14.5 g/L respectively.
Example 4
The preparation process and the operating conditions were the same as in example 1. The difference lies in that: 2g/L calcium carbonate is added into the fermentation medium, and the pH is not controlled in the whole process. Through detection, the concentration of butanol in the fermentation liquor is 8.56g/L, the concentration of isopropanol is 7.93g/L, and the concentrations of glucose and xylose are both lower than 0.1 g/L.
Example 5
The preparation process and the operating conditions were the same as in example 1. The difference lies in that: the seed culture medium adopts the following formula: the dosage of the hydrolyzed mixed sugar solution is that the concentration of sugar is 8g/L, peptone is 10g/L, yeast extract is 3g/L, beef extract is 10g/L, cysteine hydrochloride is 0.4g/L, sodium chloride is 2g/L, sodium acetate is 3g/L, the pH is adjusted to 7.5, and sterilization is carried out for 30min at 115 ℃. The detection proves that the concentration of the butanol in the fermentation liquor is 6.25g/L, the concentration of the isopropanol is 5.54g/L, and the residual glucose and the residual xylose are 4.5g/L and 7.6g/L respectively.
Example 6
The preparation process and the operating conditions were the same as in example 1. The difference lies in that: the nitrogen source in the fermentation medium is only 10g/L of yeast extract, 0.6g/L of beef extract and 1.0g/L of ammonium acetate. The detection proves that the concentration of the butanol in the fermentation liquor is 5.89g/L, the concentration of the isopropanol is 5.37g/L, and the concentrations of the residual glucose and the xylose are 5.5g/L and 8.7g/L respectively.
Example 7
The preparation process and the operating conditions were the same as in example 1. The difference lies in that: the nitrogen source in the fermentation medium is only 11g/L of soybean peptone and 0.6g/L of beef extract. The detection proves that the concentration of the butanol in the fermentation liquor is 5.87g/L, the concentration of the isopropanol is 5.26g/L, and the concentrations of the residual glucose and the xylose are 5.8 g/L and 8.4 g/L respectively.
Example 8
The preparation process and the operating conditions were the same as in example 1. The difference lies in that: the nitrogen source in the fermentation medium is only soybean peptone 11g/L and ammonium sulfate 1.0 g/L. The detection proves that the concentration of the butanol in the fermentation liquor is 5.63g/L, the concentration of the isopropanol is 5.05g/L, and the concentrations of the residual glucose and the xylose are respectively 6.8g/L and 9.0 g/L.
Example 9
The preparation process and the operating conditions were the same as in example 1. The difference lies in that: the nutrient salts supplemented in the fermentation medium are 3g/L of monopotassium phosphate, 0.3g/L of magnesium sulfate, 0.1g/L of ferrous sulfate and 0.4g/L of sodium chloride. The detection proves that the concentration of the butanol in the fermentation liquor is 5.92g/L, the concentration of the isopropanol is 5.33g/L, and the concentrations of the residual glucose and the xylose are respectively 6.3g/L and 7.8 g/L.
Example 10
The preparation process and the operating conditions were the same as in example 1. The difference lies in that: the nutrient salts supplemented in the fermentation medium are 3g/L of monopotassium phosphate, 0.1g/L of ferrous sulfate, 0.4g/L of sodium chloride and 0.1g/L of calcium chloride. The detection proves that the concentration of the butanol in the fermentation liquor is 5.87g/L, the concentration of the isopropanol is 5.24g/L, and the concentrations of the residual glucose and the xylose are 5.7g/L and 8.9g/L respectively.
Example 11
The preparation process and the operating conditions were the same as in example 1. The difference lies in that: no biotin was added to the fermentation medium. The detection proves that the concentration of the butanol in the fermentation liquor is 5.93g/L, the concentration of the isopropanol is 5.31g/L, and the concentrations of the residual glucose and the xylose are respectively 6.3g/L and 8.3 g/L.
Example 12
The preparation process and the operating conditions were the same as in example 1. The difference lies in that: the fermentation medium adopts sodium hydroxide to replace calcium hydroxide to adjust the pH. The detection proves that the concentration of the butanol in the fermentation liquor is 4.88 g/L, the concentration of the isopropanol is 4.24g/L, and the concentrations of the residual glucose and the xylose are respectively 9.5g/L and 11.6 g/L.
Example 13
The preparation process and the operating conditions were the same as in example 1. The difference lies in that: the fermentation process is anaerobic fermentation. The detection proves that the concentration of the butanol in the fermentation liquor is 5.98g/L, the concentration of the isopropanol is 5.49g/L, and the residual glucose and the residual xylose are 7.1 g/L and 5.1g/L respectively.
Comparative example 1
The preparation process and the operating conditions were the same as in example 1, except that: the corn stalk material is maintained in a detention device of a steam explosion device for 10min at the temperature of 180 ℃ and the pressure of 1.8 MPa, and is instantaneously decompressed and exploded. The thallus does not grow within 120h of fermentation, and no product is generated.
Comparative example 2
The process flow and the operating conditions were the same as in example 1, except that: using Clostridium beijerinckii described in Chinese patent 201410731297.9: (Clostridium beijerinckii) CM 20. Fermenting for 72 hours to obtain fermentation liquor containing butanol, acetone and ethanol, wherein the fermentation liquor has no glucose and xylose residue, the total solvent concentration is 15.04 g/L, the butanol concentration is 8.50g/L, the acetone concentration is 6.14g/L and the ethanol concentration is 0.40 g/L.
Claims (13)
1. A method for producing isopropanol and butanol by fermentation by using lignocellulose as a raw material is characterized by comprising the following steps: (1) performing steam explosion pretreatment on the lignocellulose raw material, wherein the steam explosion adopts neutral steam explosion pretreatment, and the method specifically comprises the following steps: pulverizing the lignocellulose raw material to 0.5-5 cm, adding tap water with the mass of 2-4 times, soaking, introducing into a detention device of a steam explosion device, maintaining for 5-20min at the temperature of 120-; (2) carrying out enzymolysis on the pretreated material to obtain a cellulose and hemicellulose hydrolyzed mixed sugar solution; (3) directly using the hydrolyzed mixed sugar solution obtained in the step (2) for preparing a fermentation culture medium without carrying out solid-liquid separation and detoxification treatment, and preparing the fermentation culture medium by taking the hydrolyzed mixed sugar solution as a carbon source and supplementing nitrogen sources, nutrient salts and vitamins; (4) clostridium beijerinckii (C., (B.))Clostridium beijerinckii) XH0906 with preservation number of CGMCC number 9124Inoculating the seed solution into the fermentation culture medium in the step (3), fermenting and producing butanol and isopropanol, wherein N does not need to be introduced in the culture process2Maintaining an anaerobic environment.
2. The method of claim 1, wherein: the lignocellulose raw material in the step (1) is straw, wood chips or energy plants containing cellulose, hemicellulose and lignin.
3. The method of claim 1, wherein: preparing the pretreated materials in the step (1) into feed liquid with a solid-to-liquid ratio of 8-20% (w/v), and then adding a cellulase preparation for enzymolysis.
4. A method according to claim 1 or 3, characterized in that: the addition amount of the cellulase in the step (2) is 20-50FPIU/g cellulose, the pH value of enzymolysis is 4.5-5.5, the temperature is 45-55 ℃, the stirring speed is 50-300r/min, and the enzymolysis time is 24-72 h.
5. The method of claim 1, wherein: the dosage of the hydrolysis mixed sugar liquid in the step (3) is to ensure that the sugar concentration in the culture medium is 30-60 g/L.
6. The method of claim 1, wherein: the nitrogen source supplemented in the fermentation medium is an organic nitrogen source or/and an inorganic nitrogen source, the organic nitrogen source is one or more of yeast extract, peptone, beef extract, corn steep liquor and soybean meal hydrolysate, and the concentration is 0.1-20 g/L; the inorganic nitrogen source is one or more of ammonium acetate, sodium nitrate and ammonium sulfate, and the concentration is 0.05-5 g/L; the supplementary nutrient salt is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, sodium hydroxide, ferrous sulfate, ferric sulfate, magnesium sulfate, calcium chloride, manganese sulfate and calcium carbonate, and the concentration is 1-6 g/L; the vitamin is one or more of vitamin B1, biotin, and p-aminobenzoic acid, and has concentration of 0.01-0.1 g/L.
7. The method of claim 6, wherein: the supplemented nitrogen source is 8-12g/L of soybean peptone, 0.4-0.8g/L of beef extract and 0.6-1.2g/L of ammonium sulfate; the supplementary nutrient salt is 2-4 g/L of monopotassium phosphate, 0.3-0.6 g/L of sodium chloride, 0.05-0.2 g/L of ferrous sulfate, 0.1-0.4g/L of magnesium sulfate and 0.05-0.2 g/L of calcium chloride.
8. The method of claim 1, 6 or 7, wherein: 0.2-5g/L calcium carbonate is added into the fermentation medium, and the pH is not controlled in the whole process.
9. The method of claim 1, wherein: the pH value of the fermentation medium in the step (3) is 6.0-8.0, and calcium hydroxide is adopted for adjustment.
10. The method of claim 1, wherein: the seed culture medium for preparing the seed liquid in the step (4) adopts the seed culture medium with the following formula: the dosage of the hydrolyzed mixed sugar solution is such that the sugar concentration is 2-8 g/L, peptone is 5-15 g/L, yeast extract is 2-5g/L, beef extract is 5-15 g/L, cysteine hydrochloride is 0.2-0.6 g/L, sodium chloride is 1-4 g/L, sodium acetate is 2-4 g/L, the pH is adjusted to 6.0-8.5, and sterilization is carried out at 115 ℃ for 30 min.
11. The method of claim 1, wherein: and (4) preparing the seed solution in the step (4), namely activating a clostridium beijerinckii XH0906 plate in a solid culture medium, placing the plate in an anaerobic environment, culturing for 12 to 48 hours at the temperature of between 28 and 42 ℃, then picking the activated single colony, inoculating the single colony into the seed culture medium, and standing and culturing for 12 to 48 hours at the temperature of between 28 and 42 ℃.
12. The method of claim 1, wherein: and (4) fermenting by adopting a stirring tank, in-situ extraction fermentation or gas in-situ extraction fermentation, wherein the stirring linear speed is 50-300 r/min.
13. The method of claim 1, wherein: and (4) inoculating the seed liquid obtained in the step (4) into a liquid fermentation culture medium by the volume ratio of 2-20%, wherein the fermentation temperature is 28-42 ℃, and the fermentation time is 24-120 h.
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